Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells

Abstract

The tumour-associated antigen mucin 1 (MUC1) is a multifunctional protein involved in protection of mucous membranes, signal transduction, and modulation of the immune system. More than 70% of cancers overexpress MUC1, making MUC1 a potential target for immunotherapy. In the present study, MUC1 transgenic mice were crossed with syngeneic strains that express the polyomavirus middle-T oncogene (PyMT) driven by the mouse mammary tumour virus promoter long-terminal repeat (MMTV-LTR). The resultant breed (MMT mice) developed spontaneous MUC1-expressing mammary carcinomas with 100% penetrance at 8-15 weeks of age. As found in human breast cancer, the mammary carcinoma in MMT mice arose in multiple stages. Immunization with fusions of dendritic cells and MUC1-positive tumour cells (FC/MUC1) induced MUC1-specific immune responses that blocked or delayed the development of spontaneous breast carcinomas. In contrast, there was no delay of tumour development in MMT mice immunized with irradiated MC38/MUC1 tumour cells. The efficacy of fusion cells was closely correlated with the timing of initial immunization. Immunization with FC/MUC1 initiated in MMT mice at < 1, 1-2 and 2-3 months of age rendered 33, 5 and 0% of mice free of tumour, respectively, up to 6 months. Whereas mice immunized in the later stage of tumour development succumbed to their disease, immunization resulted in control of tumour progression and prolongation of life. These results indicate that immunization with FC/MUC1 can generate an anti-MUC1 response that is sufficient to delay the development of spontaneous mammary carcinomas and control tumour progression in MMT mice.

Figure 1

Mammary carcinoma development in MMT and MT mice. (a) Growth rate of spontaneous mammary carcinomas in MMT (•) and MT (○) mice. Three mice were used at each time-point. The error bars represent SE of the mean. (b) Histological examination with H&E staining of spontaneous mammary tumours in MT and MMT mice at different ages (×16).

Figure 2

MUC1 expression on mammary carcinomas. (a) Lysates from MMT carcinoma (lane 1), MT carcinoma (lane 2) and human MCF-7 tumour cells (lane 3) were analysed by western immunoblotting with anti-MUC1 mAb DF3. (b) PCR was used to detect MT and MUC1 genes in mammary carcinoma from MMT and MT mice. (c, d) Immunohistochemical staining with mAb DF3 to detect the expression of MUC1 on mammary carcinomas from MT and MMT mice (×40).

Figure 3

Anti-MUC1 humoral response induced in MMT mice by FC/MUC1 immunization. (a) The measurement of a single serum sample from one female MMT mouse or her littermates. The mice at 4 weeks of age were immunized with 5 × 10⁵ FC/MUC1 cells (•), irradiated MC38/MUC1 cells (▪) or FC/MC38 cells (□), or treated with PBS (○). The immunization was repeated at 8 and 12 weeks. Mice were killed 1 week after the last immunization. Serum from the mice was collected and anti-MUC1 antibodies were detected by ELISA. The results are expressed as the mean ± SD of three replicates. (b) Serum was collected from MMT mice of various ages (three or four mice per group) that had been immunized at different times with 5 × 10⁵ FC/MUC1 cells (•) or treated with PBS (○) and assayed for the presence of anti-MUC1 antibody. Each dot represents the level of anti-MUC1 antibody (1 : 5 dilution) at the indicated age and number of immunizations. (c) MMT mice were immunized with 5 × 10⁵ FC/MUC1 cells (•) at 4 weeks of age. The immunization was repeated at 8 and 12 weeks. Their littermates were immunized with FC/MC38 cells (□), irradiated MC38/MUC1 cells (▪) or PBS (○). One week after the last immunization, the mice were killed and splenocytes were purified through nylon wool to remove antigen-presenting cells. T cells were incubated with mammary carcinoma cells from MMT mice at the indicated effector:target ratios. CTL activity was determined by the ⁵¹Cr release assay. The results are expressed as the mean ± SD of three replicates. (d) Splenocytes were isolated from MMT mice of various ages (three or four mice per group) that had been immunized at different times with 5 × 10⁵ FC/MUC1 cells (•) or PBS (○). CTL activity against mammary carcinoma cells from MMT mice at an effector:target ratio of 100 : 1 was determined by ⁵¹Cr release. Each dot represents the CTL activity at the indicated age and number of immunizations. (e) Splenocytes were isolated from MMT mice immunized five times with 5 × 10⁵ FC/MUC1 (right panel) or irradiated MC38/MUC1 tumour cells (middle panel) or treated with PBS (left panel). CTL activity against MC38 (○) and MC38/MUC1 (•), MT (□) and MMT (▪) mammary carcinoma cells, and YAC-1 (▵) cells at the indicated effector:target ratio was determined by ⁵¹Cr release.

Figure 4

Inhibition of spontaneous mammary carcinoma in vivo. (a) Schedule of immunization with FC/MUC1. Female MMT and MT mice were vaccinated subcutaneously with 5 × 10⁵ FC/MUC1 cells, FC/MC38 cells or irradiated MC38/MUC1 cells at the base of tail at < 1 month of age. Immunization was repeated for four additional times at 4-week intervals. (b) Tumour incidence in MT (left panel) and MMT (right panel) mice immunized with FC/MUC1 cells (•), FC/MC38 cells (□), irradiated MC38/MUC1 cells (▴) or PBS (○). Each dot represents the appearance of tumour for individual mice at the indicated age.

Figure 5

Immunoprevention and immunotherapy with FC/MUC1 immunization. (a) Tumour incidence in MMT mice in which immunization with FC/MUC1 cells was begun at <1 month (•), 1–2 months (▴), or over 2 months (▪). Non-vaccinated age-matched MMT mice were used as the control (□). (b) Photograph of female littermates of MMT mice at age 14 weeks treated with PBS (left panel) or at age 26 weeks treated with FC/MUC1 fusion (right panel). The mice were < 1 month old when treatment with PBS or FC/MUC1 immunization was commenced. (c, d) Photomicrograph of mammary carcinoma from a non-vaccinated MMT mouse at the age of 14 weeks (left panels) and of mammary tissue from a vaccinated MMT mouse at the age of 26 weeks (right panels). The mice were < 1 month old when treatment with PBS or immunization was commenced. The upper panels have magnifications of ×10 and the lower panels ×40.